Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.
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PMID:Amino terminal regions of poliovirus 2C protein mediate membrane binding. 774 26

During the cellular protein targeting process, zeins (maize storage proteins) are retained in the endoplasmic reticulum (ER) where they accumulate into protein bodies. There are circumstantial and preliminary data indicating that the 27K zein, a class of zein proteins, may span the ER membrane. This potential transmembrane domain is considered very significant with regard to the mechanism of ER retention for zeins. The potential transmembrane domain may permit the 27K zein to remain in the ER and to serve as an anchor for other classes of zein, thus acting as a nucleating factor for protein body formation. This study investigated the potential transmembrane feature in a heterologous system (Xenopus laevis oocyte). Following injection of synthetic 27K zein mRNA, isolated protein vesicles were subjected to proteinase K digestion, surface biotinylation and alkaline extraction. Throughout three categories of assay the possible role of the 27K zein as a transmembrane protein was consistently refuted in this study. The 27K zein polypeptide was affected by neither proteinase K digestion nor biotinylation and was released from alkali-stripped membranes. This study, therefore, concludes that the 27K zein is not a protein body nucleating factor by virtue of an ER transmembrane feature.
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PMID:Evidence against a potential endoplasmic reticulum transmembrane domain of 27K zein expressed in Xenopus oocytes. 777 Apr 58

The structure and post-translational processing of the metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was analysed by in vitro cell-free translation, protease protection and deglycosylation. We show that mGluR1 alpha can be synthesized in the rabbit-reticulocyte translation system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microsomes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide-N-glycosidase F (PNGF) demonstrated that the increase in the apparent molecular weight of the processed translation product was due to N-linked glycosylation. Addition of the non-selective protease, proteinase K; resulted in the loss of this 147 kDa band and the appearance of a protected fragment of approx 92 kDa. A carboxy-terminal deletion mutant of mGluR1 alpha was almost completely protected from protease action. These data show that the amino terminal of mGluR1 alpha is translocated into the lumen of the endoplasmic reticulum and will consequently be located extracellularly when targeted to the plasma membrane. The data presented here on mGluR1 alpha indicates the potential of in vitro translation and protease protection in the study of the molecular structure and processing of glutamate receptors.
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PMID:In vitro translation and membrane topology of rat recombinant mGluR1 alpha. 783 18

The endoplasmic reticulum (ER) not only links the translational machinery to the endomembrane system in eukaryotic cells but also provides a protective environment for the folding of exoplasmic proteins translocated across the ER membrane. Here we describe that the lumenal surface of the ER membranes transiently tethers the folding intermediate of secretory proteins via a 90-kDa ER membrane protein, calnexin. We demonstrate that p70, the precursor to gp80, the major secretory protein in Madin-Darby canine kidney (MDCK) cells, was bound transiently to calnexin in the immediate post-synthetic period (0-10 min) and showed a t1/2 for dissociation from calnexin of 2.5 min. The bound p70 was found to be incompletely folded as assessed by susceptibility to proteinase K digestion. Perturbation of the redox state by 5 mM dithiothreitol or 1 mM diamide markedly inhibited the dissociation of p70 from calnexin (t1/2 > 30 min). Cellular depletion of ATP led to premature dissociation of p70 from calnexin and the formation of p70 aggregates that did not bind calnexin. These findings demonstrate that nascent unfolded p70 is tethered to calnexin during normal protein maturation, including the formation and editing of disulfide bonds and that ATP is required for the productive interaction of gp80 and calnexin.
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PMID:Chaperone function of calnexin for the folding intermediate of gp80, the major secretory protein in MDCK cells. Regulation by redox state and ATP. 790 29

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.
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PMID:The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies. 862 35

The cDNA clone pSM/1.6 encoding the 26.5-kDa precursor molecule of the 16/17-kDa microneme antigen of Sarcocystis muris cyst merozoites was expressed in a cell-free translation/translocation system to study translocation of the protein across membranes. The antigen was found to be translocated across heterologous endoplasmic reticulum membranes. Translocation was accompanied by cleavage of a signal peptide to create a 23-kDa polypeptide that was completely protected from digestion with proteinase K. Pulse-chase analysis of [35S]-methionine-labeled S. muris cyst merozoites demonstrated that the 16/17-kDa antigen derived from a 23-kDa precursor molecule and that its processing occurred at between a few minutes and 2 h after biosynthesis. This leads to the conclusion that the native microneme antigen is secreted from the parasite cell via the endoplasmic reticulum. Sorting into micronemes might occur during transition through a Golgi-like structure, involving cleavage of the hydrophilic propeptide to create the mature 16/17-kDa protein.
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PMID:In vitro biosynthesis and in vivo processing of the major microneme antigen of Sarcocystis muris cyst merozoites. 873 88

The invariant chain (I chain) associates with major histocompatibility complex class II alphabeta heterodimers upon synthesis, preventing them from binding peptides and unfolded proteins in the endoplasmic reticulum and directing class II transport to post-Golgi endosomal compartments. To assess which regions of the I chain are involved in binding class II molecules, we have studied proteolytic fragments of the I chain generated both by natural proteolytic degradation of alphabeta dimer-invariant chain complexes (alphabeta.I) within human B cells and by in vitro digestion of purified alphabeta middle dotI complexes with proteinase K. The 18-kDa luminal I chain fragment generated by proteinase K, called K3, remains associated with alphabeta dimers and retains the complex (alphabeta.K3) in a high molecular mass nonameric configuration. The N terminus of the K3 fragment was identified as glycine 110. This indicates that the K3 fragment lies outside of the class II-associated invariant chain peptide region (amino acids 81-104) of the I chain, shown to be important for initial alphabeta.I assembly. An N-terminal 12-kDa I chain fragment called p12, generated intracellularly, was also analyzed and was found to remain associated with alphabeta dimers in a high molecular mass form analogous to the nonameric alphabeta.I complex. These results demonstrate that at least two class II contact points exist along the length of the I chain and that different regions of the I chain can stabilize the alphabeta.I nonamer. Additional evidence suggests that the O-linked glycan(s) characteristic of the I chain is added to the short C-terminal region absent from the K3 fragment.
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PMID:Trimeric interactions of the invariant chain and its association with major histocompatibility complex class II alpha beta dimers. 879 70

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.
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PMID:Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting. 928 16

Acetyl-CoA carboxylase (ACC1) catalyzes the first and rate limiting step of de novo fatty acid synthesis. Defects in Acc1p were recently correlated with an altered structure/function of the nuclear envelope in yeast. The subcellular distribution of the enzyme was determined in wild-type and mutant cells by cell fractionation and confocal immunofluorescence microscopy. Even though fatty acid synthesis is generally considered to be a cytosolic reaction, we found that Acc1p cofractionated with nuclei and the ER (endoplasmic reticulum) marker BiP/Kar2p. Membrane-bound Acc1p was susceptible to proteinase K digestion and was solubilized by mild salt treatment indicating that it is loosely associated with the cytosolic surface of the nuclear ER membrane. Consistent with these observations, immunofluorescence analysis revealed that Acc1p was distributed in a gradient within the cytoplasm that had its highest concentration around the ER. Possible association of Acc1p with the nuclear pore complexes (NPCs) was investigated in strains that display NPC clustering. Results of these experiments suggest that Acc1p localization is independent of NPC distribution. We propose that association of Acc1p with the cytoplasmic surface of the ER membrane is physiologically relevant to "channel" the enzymatic product of Acc1p, malonyl-CoA, to a putative ER-localized fatty acid chain elongase complex.
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PMID:Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum. 943 37

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.
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PMID:African swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum. 976 44


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